KR20160029734A - Detecting endocrine disrupting compounds - Google Patents

Abstract

The present invention relates to the detection of endocrine disrupting compounds. In particular, the invention provides a detection device for detecting an endocrine disruptor compound comprising a support structure and a ligand binding domain (LBD) of at least one sex hormone receptor immobilized on said support structure. The detection device further comprises linker molecules between the support structure and the ligand binding domain for immobilizing the LBD on the support structure.

Description

DETECTING ENDOCRINE DISRUPTION COMPOUNDS < RTI ID = 0.0 >

The present invention relates to the detection of endocrine disrupting compounds. In particular, the present invention relates to a detection device for detecting an endocrine disruptor compound and a method for detecting an endocrine disruptor compound.

It is well known that certain hormone activators in the environment can disturb the chemical messenger (hormones) of the endocrine system by sending false signals or blocking legitimate signals. Estimated hormone activators, also known as endocrine disrupting compounds (EDC), have a deleterious effect on humans and wildlife by mimicking, blocking and disturbing the physiological actions of hormones. The American Endocrinology Society conducted a comprehensive report in 2009 that showed evidence that endocrine disruptors affect male and female reproduction, breast development and cancer, prostate cancer, neuroendocrine activity, thyroid activity, metabolism, obesity, and cardiovascular endocrinology (Diamanti -Kandarakis et al., 2009). This report concludes that results from animal models, human clinical observations, and epidemiological studies provide overwhelming evidence that EDCs provide important concerns about public health.

Hormones interact with their corresponding receptors in target cells to exert their effects, such as growth, development, behavior and reproduction, and prompt normal biological function. As in the case of EDC, interference with the activity of hormones can lead to reversible or irreversible abnormal biological outcomes, including dwarf growth, short term memory impairment, tubal pregnancy, low sperm count, reproductive and immune system damage. As researchers continue to study the adverse effects caused by these harmful compounds on humans and wildlife, they are likely to continue to be of significant, and often permanent, effect at significantly lower doses.

Endocrine disruptors can be classified into three main groups: the androgens (compounds that mimic or block natural testosterone), thyroid (compounds that have direct or indirect effects on the thyroid), and estrogen Or blocking compounds). Despite the broad spectrum of EDC, estrogen compounds (ECs) are the most promising and are being studied for these compounds. This may be all due to the importance of EC in cancer research specifically related to female sexual development and disability (McLachlan et al., 2006; Darbre and Charles, 2010; Fenton, 2006; Newbold et al., 2007). Estrogen compounds are found in low doses in thousands of products and include compounds such as diethylstilbestrol, bisphenol A, polybrominated diphenyl ether and phthalate. Androgen EDCs have attracted much attention due to their effects on vigor and fertility (Bay, Asklund et al., 2006; Sharpe, 2006; Skakkebak et al., 2001). Endocrine disrupting compounds have been widely reported to exist in very low concentrations in the environment but their relatively high fat solubility allows them to bioaccumulate in local deposits of organisms and animals at high levels in the food chain, Resulting in a remarkable physiological response. Other relevant sources of EDC are found in industrial chemicals such as pesticides, herbicides, fungicides, plasticizers, plastics, resins and detergents (Diamanti-Kandarakis et al., 2009). The hydrophobicity of EDC combined with other chemical properties presents a unique challenge to environmental analysis chemists in the development of techniques necessary for EDC detection and screening. Several analytical techniques are used. These methods include solid phase extraction (SPE) followed by high performance liquid chromatography (HPLC), liquid chromatography / weight spectrophotometer (LC / MS) or gas chromatography / weight spectrophotometer (GC / MS). However, these techniques are limited to general EDC monitoring, due to relatively high instrument cost, labor intensive and, in some cases, relatively poor sensitivity. Advanced analytical procedures are usually only specific for a single analyte, or a structurally related limited class of compounds.

Affinity chromatography (AC) is a powerful chromatographic technique that utilizes specific interactions between biomolecules and ligands (hormone receptors and hormones) that affect the specific binding and isolation of ligands or substrate analogs (Cuatrecasas, 1970) . During AC, biospecific and reversible interactions are used for selective separation and purification of biomolecules from complex biological matrices. These systems are increasingly being applied in the biotechnology field due to their ability to specifically bind and remove biomolecules from complex mixtures. A typical affinity system consists of two distinct parts: a mobile phase carrying biomolecules to be separated, and a solid phase usually transformed to carry affinity ligands. Despite the widespread use of these systems, they still have some disadvantages, such as requiring large column set-up and longer diffusion path lengths, Resulting in a significant increase in the time required for the entire downstream process. Membrane affinity chromatography (MAC) was introduced to overcome the major drawbacks of column affinity chromatography. Its introduction significantly reduces the number of steps required to obtain a pure product due to the specificity of the interaction between the stationary phase and the target biomolecule, and provides a larger surface area and a shorter diffusion path length provided by the system. Because of the advantages described above, the affinity membrane system (AMS) could act as a powerful method for the process of analytically detecting and possibly eliminating EDC from the environment.

Estrogen compounds are EDCs that mimic or block endogenous estrogenic activity by binding to the ligand binding domain (hER < alpha > LBD) of the estrogen receptor (ER) in the endocrine system. By using the interaction between estrogens and their receptors and using the chemical information obtained from these interactions, a more secure and specific analytical functionalized affinity membrane system for early capture, enrichment and quantitative detection of EC was developed . This method will be assisted and used in combination with advanced technique analysis techniques such as HPLC, GC / MS, and LC / MS to detect EC in the environment. Similar systems for androgen EDC using hARLBD can also be used.

The present inventors have identified new detection and detection procedures that are rapid and effective in monitoring and detecting endocrine disrupting compounds.

Reference herein to the term " sex hormone receptor "refers in particular to the estrogen and androgen receptor.

The following abbreviations will be used herein:

³ HE2 2, 4, 6, 7 ³ H 17? -Estradiol

AC Affinity chromatography

AMS Affinity Membrane System

AR Androgen receptor

CA Cellulose acetate

CPM Count per minute

DMDDO Dicarboxy methyl-3,6-diazaoctanedioate

DMF Dimethylformamide

DMSO Dimethylsulfoxide

E2 17β-estradiol

EC Estrogen compound

EDC Endocrine disruptor compound

EDTA Ethyldiamine tetraacetic acid

ELISA Enzyme-linked immunosorbent assay

ELRA Enzyme bound receptor < RTI ID = 0.0 >

ER Estrogen receptor

ERE Estrogen response element

FT-IR Porery Transfection Infrared Spectrometer

GC Gas chromatography

GC / MS Gas Chromatography / Weight Spectrophotometer

HF Hollow fiber

HFF Hollow fine fibers

His ₆ Hexahistidine

HPLC High Performance Liquid Chromatography

HRP Horseradish peroxidase < RTI ID = 0.0 >

MAC Immobilized metal ion affinity chromatography

LBD The ligand binding domain (ligand binding domain)

LC / MS Liquid chromatography / weight spectrophotometer

LLD Lowest detection limit

MAC Membrane affinity chromatography

MBP Maltose binding protein

MS Weight spectrophotometer

NMR Nuclear magnetic resonance

PCR Polymerase chain reaction

PDMDDO Pluronic-N, N-dicarboxymethyl-3,6-diazaoctanedioate

PEI Polyetherimide

PIXES Proton-induced x-ray emission spectrophotometer

PS polystyrene

PSU Polysulfone

PVDF Polyvinylidene fluoride

SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM Scanning electron microscope

SPE Solid-phase extraction

UF Ultrafiltration

Reference will be made to the following references in the present specification:

BAY, K., ASKLUND, C, SKAKKEBAEK, N.E. and ANDERSSON, A. M., 2006. Testicular dysgenesis syndrome: possible role of endocrine disrupters. Best Practice & Research in Clinical Endocrinology & Metabolism, 20 (1), 77-90.

CUATRECASAS, P., 1970. Protein Purification by affinity chromatography. Journal of Biological Chemistry, 245 (12), 3059-3065.

DARBRE, P.D. and CHARLES, A.K., 2010. Environmental oestrogens and breast cancer: evidence for combined involvement of dietary, household and cosmetic xenoestrogens. Anticancer Research, 30 (3), 815-827.

DIAMANTI-KANDARAKIS, E., BOURGUIGNON, J. P., GIUDICE, L. C., HAUSER, R., PRINS, G. S., SOTO, A. M., ZOELLER, R.T. and GORE, A.C., 2009. Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocrine Reviews, 30 (4), 293-342.

FENTON, S.E., 2006. Endocrine-disrupting compounds and mammary gland development: early exposure and later life consequences. Endocrinology 147 (6) (Supplement): S18-S24.

L. and SWART, P., 2006. A Pluronic-coupled metal-chelating ligand for membrane affinity chromatography. Journal of Membrane Science, 279 (1-2), 120-128.

MCLACHLAN, J. A., SIMPSON, E. and MARTIN, M., 2006. Endocrine disrupters and female reproductive health. Best Practice & Research Clinical Endocrinology & Metabolism, 20 (1), 63-75.

NEWBOLD, R.R., JEFFERSON, W.N. and PADILLA-BANKS, E., 2007. Long-term adverse effects of neonatal exposure to bisphenolate on the murine female reproductive tract. Reproductive Toxicology, 24 (2), 253-258.

SHARPE, R.M., 2006. Pathways of endocrine disruption during male sexual differentiation and masculinisation. Best Practice & Research Clinical Endocrinology & Metabolism, 20 (1), 91-110.

SKAKKEBAK, N., RAJPERT-DE MEYTS, E. and MAIN, K., 2001. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects: Opinion. Human Reproduction, 16 (5), 972-978.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention,

Support structure; And

There is provided a detection device for detecting an endocrine disruptor compound, comprising a ligand binding domain (LBD) of at least one sex hormone receptor immobilized on said support structure.

The detection device may comprise linker molecules between the support structure and the ligand binding domain.

The endocrine disrupting compound may be in the form of one or more androgen and estrogen compounds.

The sex hormone receptor may be in the form of one or more estrogen receptors and androgen receptors. In particular, the sex hormone receptor may be in the form of one or more human estrogen receptor and human androgen receptor having ligand binding domains hER [alpha] LBD and hARLBD, respectively. The binding domain may comprise a tag peptide (peptide tag) In his ₆ - may be in the form hARLBD - hERαLBD his or _6. Alternatively, the ligand binding domain may be expressed as a fusion protein and may be in the form of a maltose binding protein-hER alpha LBD (MBP-hER alpha LBD).

The ligand binding domain of the sex hormone receptor can be produced by cloning a gene encoding a ligand binding domain and expressing the gene in a host. Preferably, the host is Escherichia It can be coli).

The support structure may be in the form of a membrane. The support structure may be in the form of a membrane contactor (strip matrix).

In one embodiment, the membrane contactor may be in the form of a cellulose acetate hybrid membrane, particularly an affinity cellulose acetate-amylose hybrid membrane. In such embodiments, the linker molecule may be in the form of a maltose binding protein.

In another embodiment, the support structure may be in the form of an inert film. The inert film may be in the form of a synthetic polymer film. The synthetic polymer film can be produced using an immersion precipitation technique. The synthetic polymer membrane may be planar and non-porous. The synthetic polymer film may be cast from one or more polysulfone (PSU), polyether imide (PEI), and polyvinylidene fluoride (PVDF) solutions.

In such embodiments, the linker molecule may be in the form of a modified poloxamer. The poloxamer may be in the form of a bi-functional block copolymer surfactant end-terminated with a primary hydroxyl group. The poloxamer may be in the form of poly (ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol) (hereinafter also referred to as Pluronic F108, also known as PEG-PPG-PEG).

The modified Pluronic F108 can be in the form of Pluronic F108, in which hydroxyl end groups can be modified to metal chelating sites to specifically bind Ni-ions and serve as an immobilized metal-displaced chromatography matrix, and metal- Lt; RTI ID = 0.0 > ligand < / RTI > binding domain. In particular, ethylenediaminetetraacetic acid dianhydride is coupled to the terminal hydroxyl end group of Pluronic F108 via a two step reaction to form a new metal affinity ligand, Pluronic-N, N-dicarboxymethyl-3,6-diazooctanedioate Pluronic-DMDDO).

In another aspect of the present invention,

Contacting a detection device as described above with a sample to be tested such that if the sample contains an endocrine disruptor compound, allowing the compound to bind to the ligand binding domain; And

And performing a colorimetric assay to display the combined endocrine disrupting compound. ≪ Desc / Clms Page number 3 >

The sample to be tested may be in the form of a water sample.

The step of bringing the detection device into contact with the sample may include a method of submerging the detection device into the sample, dropping the sample on the detection device, dipping the detection device into a sample or the like can do.

In one embodiment, the practice of a colorimetric assay may include,

Saturating the receptor with EDC to cause the receptor to change shape and activate;

Adding a monoclonal antibody produced against the activated receptor and bound to an enzyme to cause the antibody-enzyme complex to bind to the activated binding receptor;

Observing the bound enzyme to show the presence of EDC in the sample;

. ≪ / RTI >

In such embodiments, the enzyme may be in the form of an enzyme that produces a colorimetric, fluorescent or luminescent derivative of the activated binding receptor. The enzyme may be in the form of a horseradish peroxidase.

Implementation of a colorimetric essay in another embodiment,

Adding an enzyme labeled steroid ligand to a specific receptor; And

Determining the degree of receptor saturation;

. ≪ / RTI >

In another embodiment, the practice of a colorimetric assay is performed by,

Adding a heat shock protein that binds and activates a coupled receptor; And

Adding an antibody to the heat shock protein to display bound receptors to show endocrine disruptor compound binding;

. ≪ / RTI >

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be described by way of embodiments with reference to the following drawings.

Figure 1 schematically shows the immobilized steroid ligand binding domain for affinity capture of EDC in drinking water;
Figure 2 schematically shows a method for detecting combined EDC.

Embodiment of the Invention

Figure 1 schematically shows a detection device for detecting an endocrine disruptor compound. The detection device comprises a support structure in the form of a membrane contactor and a ligand binding domain (LBD) of at least one sex hormone receptor immobilized on the support structure. The detection device further comprises a linker molecule in the form of Pluronic MDDO modified between the support structure and the ligand binding domain.

The detection device serves the following principle: The ligand binding domains of hER? LBD and hARLBD are immobilized on a membrane contactor matrix using non-covalent AC technology. The immobilized receptor is exposed to water containing low levels of estrogens and / or androgen compounds. These compounds bind to the corresponding receptors and are concentrated on the contactor surface through receptor ligand interactions.

Figure 2 shows a method for detecting an endocrine disruptor compound. The first step of the method involves contacting the detection device as described above with a sample to be tested and, if the sample contains an endocrine disrupting compound, causing the compound to bind to the ligand binding domain. The second step represents a combined endocrine disruptor compound by performing a colorimetric assay.

As shown in FIG. 2, upon activation, at slight increase in temperature, the hERαLBD- or hARLBD estrogen / androgen compound-complex may be expressed using a specific antibody in an enzyme-linked immuno-essay system. The presence of an estrogen / androgen compound will be indicated by the color development of a specific color on the contactor "strip ".

Way

Film making

The planar nonporous membranes were cast from a suitable solution, polysulfone (PSU), polyether imide (PEI) and polyvinylidene fluoride (PVDF). The solution was degassed prior to use to cast a 200 micron planar membrane. Non-porous hollow fiber (HF) and hollow fine fiber (HFF) membranes and externally unskinned ultrafiltration (UF) membranes were prepared by a dry-wet spinning process available from the Institute of Polymer Science, Stellenbosch University Was prepared by phase reversal method.

Amylose functionalized cellulose acetate (CA) membranes were prepared by immersion precipitation from CA and amylose solutions in DMSO. The casting mixture was used to cast the flat sheet membrane by immersion precipitation using water. The membranes were thoroughly washed with water and stored in sodium azide solution until needed. Surface membrane topography, thickness and functional strain were determined using SEM and FT-IR, respectively.

Pluronic® F108 modified linker was synthesized as follows: A tetradentate DMDDO-type ligand was obtained at the hydroxyl end of Pluronic by modifying the terminal hydroxyl group of Pluronic in a two-step reaction. This reaction was carried out by dissolving in a 10-fold excess of EDTA dianhydride and imidazole-dried DMF, followed by the addition of Pluronic F108 and reaction at 40 ° C for 8 hours. Methanol was then added and the reaction was allowed to proceed for another 8 hours before the DMF was removed in vacuo. The residue was treated with toluene to selectively dissolve the ligand-modified-Pluronic from the DMDDO by-product. The final products were mainly characterized by nuclear magnetic resonance (NMR) with the help of model ligands based on mono and diethylene glycol (Govender et al., 2006).

Receptor expression

Introduction of peptide tags, or low molecular weight proteins, to the N- or C-terminus of the protein has become a convenient and easy method for protein purification via affinity chromatography. Examples of tags include polyhistidine tags and maltose to produce fusion proteins. Because the principle of non-covalent immobilization of cleaved steroid receptors forms the basis for the test strips proposed in this scheme, the histidine tag allows the purification of the receptor and a non-covalent immobilization method according to the IMAC principle. Thus, the ligand binding domain of the steroid receptor is subcloned into a suitable vector encoding a histidine tag, and the plasmid preparation must be transformed into a suitable host organism and finally expressed a histidine tagged fusion protein.

A gene encoding the hexa-histidine-tagged hERαLBD subcloned into pET15b (a plasmid encoding the N-terminal hexahistidine tag) was obtained from the University of Illinois at Urbana-Champaign. The hERαLBD protein was expressed in Escherichia coli BL21 (DE3) pLysS. The initial yield of soluble protein compared to insoluble protein was insufficient. Prior to purification, experiments were conducted to improve the solubilization of the hERαLBD protein. These include the use of the detergent Nonidet P-40 (NP-40), which increases protein solubility during cell lysis. In addition, as shown in previous studies, 17p-estradiol (E2) and sucrose were included during the expression experiments, and these additives increased the yield of soluble protein to insoluble protein. Protein detection methods (sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blot analysis) show that an increase in soluble hERαLBD protein is achieved in the presence of 10 μM E2 (compared to expression without E2 Expression in the presence of E2 showed a higher soluble protein).

Using the full length AR gene, the truncated human androgen receptor ligand binding domain, hARLBD, was amplified by polymerase chain reaction (PCR) and subcloned into pTrcHis, a plasmid encoding the N-terminal hexahistidine tag. This preparation is indicated by AR-pTrcHis. Then, TOP10 E. coli was transformed using the above preparation. Expression and purification of hARLBD were identical to expression and purification of hER [alpha] LBD except that 17p-estradiol was absent.

In this study, the desired protein hERαLBD was expressed as a fusion protein for maltose binding protein (MBP). Maltose binding protein, a major source of carbon for gram negative bacteria, is known to have high affinity for maltose and high affinity for amylose in the absence of maltose. The affinity chromatographic column used in this study was prepared using amylose resin. Purification of the recombinant protein is possible because these proteins will bind (via MBP) to the amylose column through affinity interactions. The gene was ligated to the plasmid expression vector pMalc2 and transformed into expression E. coli TB1 cells. hERαLBD was expressed as a fusion protein for fusion partner MBP, a MalE gene with a molecular weight of 40.6 kDa.

Receptor Purification and Antibody Generation to Fused Receptors

A preliminary purification of the hER [alpha] LBD protein using a pre-packed nickel agarose column (His SpinTrap, GE Healthcare) showed an effective binding of the histidine tagged hER [alpha] LBD protein to the resin. The protein was then purified on a preparative AKTA system. hARLBD was purified under the same conditions as the purification of hER [alpha] LBD. The MBP-hER [alpha] LBD fusion protein and MBP protein were purified on an amylose column by affinity chromatography. The bound fusion protein was eluted from the affinity column by containing 10 mM maltose in elution buffer.

Purified hER [alpha] LBD, saturated with E2, was inoculated into subsequent rabbits to generate polyclonal antibodies to the E2-linked form of hER [alpha] LBD. Blood samples were taken from the rabbits on days 0 and 28. The blood samples were stored and flocculated at < RTI ID = 0.0 > 4 C < / RTI > and then centrifuged to separate serum from the agglutinates. The serum was used as an antibody source.

Bonding study

For estrogen binding studies, PVDF membranes and polystyrene microtitre plates (Immunosorp Nunc) were used as scaffolds for Pluronic-DMDDO. In addition to the hydrophobic nature of the polystyrene polymers from which they are made, the microtiter plates are empirically more practical, easier to handle, and allow higher throughput. The wells of the polystyrene plates were incubated with the Pluronic-DMDDO solution. The wells were also incubated with a Pluronic F108 solution. The Pluronic DMDDO-well was charged with Ni ^{+ 2.} The purified hERαLBD solution, which was dialyzed overnight against PBS buffer to remove E2 from the (LBD) binding site, was added to the modified and unmodified Pluronic coated wells. Uncoated wells were also coated with purified hER [alpha] LBD solution. The wells were incubated with hER [alpha] LBD solution. The working solution of 2, 4, 6, 7 ³ H-estradiol ( ³ HE2) was pipetted into each well containing modified or unmodified Pluronic, or hERαLBD. A tritiated solution of estradiol was added to the polystyrene wells containing no additives. The wells were washed with distilled water to remove excess radioactively labeled estradiol. Elution buffer (according to the washing buffer used in the purification procedure containing 500 mM imidazole) was added to each well. Each sample of 80 μL volume was delivered to a separate scintillation tube. The radioactivity of each sample was measured over 5 minutes in counts per minute (CPM) through a scintillation counter (Tri-Carb®, 1900A). A second experiment was conducted using his _6- ARLBD and tritiated testosterone. These experimental variables were exactly the same as in the hERαLBD / 3HE2 experiment.

The maltose binding protein-hER [alpha] LBD fusion protein was immobilized on an amylose functionalized membrane with amylose present on the membrane surface that provides a binding site for MBP-hER [alpha] LBD immobilization because MBP has affinity for amylose. The use of a membrane system for the selective recovery of estradiol from solution is also included herein.

Results and discussion

Membrane fabrication

Synthetic polymer membranes were prepared using immersion precipitation techniques to produce non-porous planar and capillary membranes with reproducible physical and chemical composition. The surface chemistry of the membrane polymer was confirmed using photoacoustic FT-IR analysis. Surface hydrophobicity was calculated using static and dynamic contact angle analysis for each of the planar and capillary membranes. The candidate films selected in this study add a unique rough surface to the use of the fabrication conditions. However, film surface roughness was found to decrease with 5 mg / ml Pluronic F108 after surface modification. The membrane surface hydrophobicity was in the order of PVDF>PSU> PEI. The imidazole was used to activate and dissolve the EDTA-dianhydride. This resulted in ligand attachments useful for hydrophobic surface interactions and tetradentate ligands having coordination sites on the open octahedral system for the non-polar center block. The ligand Pluronic-DMDDO is characterized using a ¹³ C NMR spectrometer and is soluble in aqueous organic solvents and can be stored indefinitely in solution or in dry conditions.

The CA / amylose mixture was successfully used as a casting dope to produce a CA / amylose functionalized membrane using the immersion precipitation technique. Membrane topography and morphology were studied using SEM techniques, while membrane surface chemistry was monitored by FT-IR. Following the information obtained from membrane morphology and surface chemistry, the membrane could be used for affinity immobilization of specific bio-ligand MBP and MBP-hER alpha LBD fusion proteins. Membranes containing a high percentage of amylose were ductile and fragile and could not be used as a solid support for subsequent experimental assays. Thus, in the present study, only 2% amylose membranes were used in the immobilization studies. However, in the future, a higher percentage of amylose may be included in the CA membrane in the future, but this will be done by using some plasticizer which will make the membrane more resistant to physical damage and deformation.

Receptor expression

The hER [alpha] LBD insert was found to be present in pET15b as shown through restriction digestion performed on isolated plasmid DNA. SDS PAGE and Western blot analysis confirmed that hER [alpha] LBD was expressed and was present in the supernatant of both medium containing the additives (E2 and sucrose) and medium without the additives. The resuspended pellets contained hER [alpha] LBD, but most hER [alpha] LBD remained in solution, hence in the supernatant.

The LBD of AR which was isolated and amplified by PCR was successfully subcloned into the pTrcHis plasmid. This plasmid construct, designated AR-pTrcHis, was used to transform TOP10 E. coli. Expression of histidine tagged ARLBD was confirmed by SDS PAGE and Western blot analysis.

This study showed that MBP-hERαLBD ligated to pMalc2 and the MBP gene (MalE product) from pMalc2 could be expressed at a high yield using the E. coli expression system. Results were obtained by SDS PAGE and western blot analysis of two cell lysates. The band corresponding to an apparent molecular weight of 66 KDa was in good agreement with the molecular weight of MBP-hERαLBD reported in the literature.

Receptor Purification and Antibody Generation to Fused Receptors

hERαLBD and ARLBD were purified on an AKTA® protein purification system. A one-step affinity purification system with amylose as a solid phase was used for the effective purification of the MBP-hER alpha LBD protein. Antibodies were generated for the estrogen-linked and unconjugated forms of hER [alpha] LBD. Higher serum dilution (> 10 ^{- 2)} between the E2 when bound form and the unbound form hERαLBD, there is a difference. Sensitivity is reduced at low serum dilutions (between 10 ^-1 and 10 ^-2 ) and primary antibodies can not be distinguished between the two forms of hERαLBD.

Bonding study

Initial testing was performed on polystyrene microtiter plates. Pluronic-DMDDO filled with hER alpha LBD was coated on the surface of a polystyrene microtiter plate. The two controls used in the experiments included wells coated with Pluronic F108 and uncoated wells. Significantly higher binding of radioactive estrogen could be seen in wells coated with Pluronic-DMDDO filled with hER [alpha] LBD compared to the control (well coated with Pluronic F108 and only uncoated wells), indicating immobilization of hER [alpha] LBD. The second Pluronic-DMDDO experiment with his ₆ -ARLBD showed similar results in that the protein-filled wells were able to bind to a significantly higher amount of radioligand compared to the control. These experiments clearly demonstrate the notion that the LBD of androgens and estrogen receptors are immobilized as well as that the receptors retain bioactivity by binding to androgens and estrogen compounds in aqueous solution, respectively.

Prior to CA / amylose flat sheet membrane binding assay, the activity and binding of the recombinant protein, MBP-hER [alpha] LBD, to the resin was tested using ³ HE2. To determine the E2-binding, the slurry of amylose resin was incubated overnight with extracts containing MBP-hER [alpha] LBD. The washed slurry was then incubated for 3 hours at room temperature with 300 [mu] L of Buffer A containing ³ HE2 working solution. The rinsing step was repeated and another 4 mL of scintillation cocktail was added to the slurry. The mixture was transferred to a scintillation vial and counted using a liquid scintillation counter.

conclusion

The present inventors have found that the present invention provides a novel detection device for detecting endocrine disrupting compounds and is capable of affinity immobilization on an inert membrane contactor of estrogen and androgenic compounds via LBD of human estrogen and androgen receptors, Of the total population.

Claims (35)

Support structure; And
A ligand binding domain (LBD) of at least one sex hormone receptor, immobilized on said support structure,
A detection device for detecting an endocrine disruptor compound. 2. The detection device of claim 1, wherein the detection device comprises linker molecules between the support structure and the ligand binding domain for immobilizing LBD on the support structure. The detection device according to claim 1, wherein the endocrine disruptor compound is either or both of an androgen and an estrogen compound. 4. The method of claim 3, wherein the sex hormone receptor is one or both of an estrogen receptor and an androgen receptor. 5. The detection device according to claim 4, wherein the sex hormone receptor is either or both of a human estrogen receptor and a human androgen receptor each having a ligand binding domain hER? LBD and hARLBD. _{6. The} detection device of claim 5, wherein the ligand binding domain comprises a peptide tag and is in the form of either his _6- hER [alpha] LBD and his ₆ -hARLBD. 6. The detection device according to claim 5, wherein the ligand binding domain hER? LBD is expressed as a fusion protein and is in the form of a maltose binding protein-hER? LBD (MBP-hER? LBD). 2. The detection device of claim 1, wherein the ligand binding domain of the at least one sex hormone receptor is prepared by cloning a gene encoding the ligand binding domain and expressing the gene in a host. 9. The detection device according to claim 8, wherein the host is E. coli. 3. The detection device according to claim 2, wherein the support structure is in the form of a membrane. 3. The detection device according to claim 2, wherein the support structure is in the form of a membrane contactor (strip matrix). 12. The detection device according to claim 11, wherein the membrane contactor is in the form of a cellulose acetate hybrid membrane. 13. The detection device according to claim 12, wherein the cellulose acetate hybrid membrane is in the form of an affinity cellulose acetate-amylose hybrid membrane. 14. The detection device of claim 13, wherein the linker molecule is in the form of a maltose binding protein. 11. The detection device according to claim 10, wherein the support structure is in the form of an inert film. 16. The detection device according to claim 15, wherein the inert film is in the form of a synthetic polymer film. 17. The detection device according to claim 16, wherein the synthetic polymer film is fabricated using an immersion precipitation technique. 17. The detection device according to claim 16, wherein the synthetic polymer membrane is planar and non-porous. 17. The detection device of claim 16, wherein the synthetic polymeric film is cast from one or more polysulfone (PSU), polyether imide (PEI), and polyvinylidene fluoride (PVDF) solutions. 20. The detection device of claim 19 wherein the linker molecule is in the modified poloxamer form. 21. The detection device of claim 20, wherein the poloxamer is in the form of a difunctional block copolymer surfactant ending with a primary hydroxyl group end. 22. The detection device of claim 21, wherein the poloxamer is in the form of a poly (ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol) (referred to as Pluronic F108). The modified Pluronic F108 of claim 22, wherein the modified Pluronic F108 is a Pluronic F108 form capable of specifically binding Ni-ions to a metal chelating site and capable of acting as an immobilized metal-displaced chromatography matrix And provides a metal affinity immobilized receptor ligand binding domain. 24. The method of claim 23, wherein ethylenediaminetetraacetic acid dianhydride is coupled to the terminal hydroxyl end group of Pluronic F108 via a two step reaction to form a novel metal affinity ligand, Pluronic-N, N- dicarboxymethyl-3,6- Diazaoctanedioate (Pluronic-DMDDO). Contacting the detection device according to any one of claims 1 to 24 with a sample to be tested such that if the sample contains an endocrine disruptor compound, allowing the compound to bind to the ligand binding domain; And
A colorimetric assay is performed to show the combined endocrine disruptor compound. 26. The method of claim 25, wherein the sample to be tested is in the form of a water sample. 27. The method according to claim 26, wherein the step of bringing the detection device into contact with the sample comprises the steps of: submerging the detection device into the sample; dropping the sample onto the detection device; or dipping the detection device into a sample or the like RTI ID = 0.0 > endocrine < / RTI > 27. The method of claim 26,
Saturating the ligand binding domain with an EDC (endocrine disruptor compound) so that the ligand binding domain is altered and activated to form an activated EDC-ligand binding domain complex;
Adding a monoclonal antibody raised against the activated EDC-ligand binding domain complex to bind the monoclonal antibody to the activated EDC-ligand binding domain complex;
Observing the bound enzyme to show the presence of EDC in the sample;
RTI ID = 0.0 > 1, < / RTI > 29. The method of claim 28, wherein the enzyme is in the form of an enzyme that produces a colorimetric, fluorescent or luminescent derivative of an activated binding receptor. 30. The method of claim 29, wherein the enzyme is in the horseradish peroxidase form. 27. The method of claim 26,
Adding an enzyme labeled steroid ligand to a specific receptor; And
Determining the degree of receptor saturation;
RTI ID = 0.0 > 1, < / RTI > 27. The method of claim 26,
Adding a heat shock protein that binds and activates a coupled receptor; And
Adding an antibody to the heat shock protein to display a bound receptor to cause endocrine disruptor compound binding
A method for detecting an endocrine disruptor compound. The detection device of claim 1, substantially for detecting an endocrine disruptor compound as described and illustrated herein. 26. The method of claim 25, substantially detecting an endocrine disruptor compound as described and exemplified herein. A detection device for detecting novel endocrine disruptor compounds substantially as herein described, and a method for detecting novel endocrine disruptor compounds.

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